The leading cause of death in Japan is malignant neoplasm, while the second leading cause is heart disease and the third leading cause is cerebrovascular disease. The majority of heart disease and cerebrovascular disease is accounted for by thrombosis, such as myocardial infarction and cerebral infarction, and together the two of these rival the number of deaths from cancer. The prevention and treatment of thrombosis continues to be an important issue. Controlling platelet activation is crucial for the prevention and treatment of thrombosis, and antiplatelet drugs are aggressively prescribed. However, the clinical efficacy have by no means come up to satisfactory levels.
Platelet function can be divided into two reactions: adhesion to subendothelial tissue at a site of vascular injury, and aggregation, in which the platelets adhere to one another. In particular, the latter is accompanied by the release of the contents of granules that are present in platelets. In addition, various molecules participate in the expression of function in the platelet activation process, and these are utilized as platelet activation markers. The effective utilization of these markers not only enables greater clarity in the diagnosis of abnormal thrombosis, but also enables a more appropriate selection of the treatment protocol.
The determination of β-thromboglobulin and platelet factor 4, which are stored in the α-granules of platelets, has been carried out in the past as an indicator of platelet activation. However, these require consideration from the influence of the blood sampling technique and the fact that they assume high values when renal function declines, and thus they are not always categorized as an appropriate markers. Moreover, in the case of high value of both β-thromboglobulin and platelet factor 4, it must be treated with caution due to the high possibility of change induced by in vitro platelet activation. Therefore, there has been desire for the appearance of a more sensitive and specific platelet activation marker.
The detection of P-selectin as a platelet activation marker has been used in recent years (Shattil S. J. et al., Blood, Volume 70 (1987), pp. 307-315; Murakami T. et al., Eur. J. Clin. Invest., Volume 26 (1996), pp. 996-1003). P-selectin is a membrane protein existing in the α-granule membrane of platelets and in the Weibel-Palade bodies of vascular endothelial cells; it migrates to the cell surface when these cells are suffered from a stimulus. P-selectin is currently categorized into two types, a membrane bound type and a soluble type. Membrane-bound P-selectin is directly detected using a fluorescence-labeled anti-P-selectin antibody and flow cytometry. However, sample handling can cause a pseudo-positive result for the expression of P-selectin in platelets, making it difficult to obtain consistent results. Soluble P-selectin, on the other hand, can be detected by sandwich ELISA. However, because P-selectin originates in platelets and vascular endothelial cells, the problem of its origin arises when the soluble P-selectin in plasma is measured.
Microscopic membrane vesicles are formed when platelets undergo activation under a variety of circumstances or are suffered from a physical stimulus; these membrane vesicles are called microparticles. Platelet microparticles are generally measured by flow cytometry, and this is important for comprehending the pathological condition of various thrombotic diseases. However, a number of problems have been noted here, such as (1) differences in sensitivity between instruments, (2) how the microparticle region is gated, (3) the fact that detection limit is near the size of the microparticles, and (4) differences between facilities based on the antibody used, and standardization of the measurement method has not been achieved.
The glycoprotein VI (GPVI) present on the platelet membrane is a collagen receptor of platelets, and it has been shown to have a central role in collagen-induced platelet activation (refer to Takayama H. Japanese Journal of Thrombosis and Hemostasis, Volume 14 (2003), Number 2, pp. 75-81). In addition, anti-mouse GPVI antibody has been reported to exhibit an antithrombotic activity by specifically inhibiting collagen-induced platelet aggregation without remarkable prolongation of bleeding time (refer to Nieswandt B. et al., J. Exp. Med., Volume 194 (2001), Number 4, pp. 459-469). It has also been reported that anti-human GPVI antibody exhibits an antithrombotic activity without causing a remarkable prolongation of bleeding time (Takayama H. et al., Blood, Volume 106 (2005), Number 11, p. 612a; WO 2005/111083 A2). Accordingly, there is an expectation that a drug that specifically inhibits collagen-mediated platelet aggregation, for example, an anti-GPVI antibody, could be a safe and highly effective antiplatelet drug.
It has been reported that the GPVI present on platelets can be detected by western blotting (Tsuji M. et al., J. Biol. Chem., Volume 272 (1997), Number 38, pp. 23528-23531). In addition, it has been confirmed by western blotting that shedding of GPVI occurs when anti-GPVI antibody is added to platelets and that soluble GPVI (sGPVI) appears in the culture supernatant (Bergmeier W. et al., Thromb. Haemost., Volume 91 (2004), pp. 951-958). It has been suggested that this shedding of sGPVI is due to the action of a metalloprotease. However, this phenomenon was artificially induced ex vivo under conditions that can almost never occur physiologically, and it was completely unclear as to whether the shedding of platelet GPVI is actually occurred in vivo, under physiological or pathological conditions. Moreover, the detection of platelet GPVI and sGPVI by western blotting is itself encumbered by a number of problems with regard to its generalization into the clinical setting, such as preparation of the sample for testing and detection sensitivity. Furthermore, the number of GPVI expressed on the platelet surface is presumed to be extremely small at approximately 3000 per platelet. Thus, in order to diagnose thrombosis/embolism, the development of a simpler test method that exhibits a high specificity and a high detection sensitivity is desired.